The recent clinical implication of efflux pump mechanisms in multi-drug resistance (MDR) warrants the identification and development of efficacious and safe agents capable of blocking efflux activity. Recent solving of the E. coli AcrB 3D structure has opened a unique opportunity to apply structural methods to discover novel efflux pump inhibitors (EPIs). The homologous tripartite MexXY-OprM efflux pump is implicated in acquired, intrinsic, and adaptive resistance to aminoglycoside (AG) antibiotics in P. aeruginosa. AGs are very potent, rapidly cidal agents that are among very few therapies still available for serious infections caused by Pseudomonas. Because EPIs reduce the amount of antibiotic required, in addition to reversing efflux-mediated acquired resistance, co-administering an EPI may also decrease the well-known toxicity associated with AG therapy. To discover novel classes of EPIs against MexXY-OprM, the aminoglycoside binding pocket MexY will be modeled in comparison to AcrB, confirmed by mutagenesis, and used for in silico high-throughput docking of approximately 1 million commercially available diverse synthetic compounds. A battery of secondary assays will allow rapid identification of EPIs among obtained virtual hits. By the end of Phase I, several distinct chemotypes will have been identified. Phase II studies will further optimize these compounds to develop a drug that will improve, preserve and expand the clinical effectiveness of AG antibiotics.